Riboswitches are elements of RNA regulation found in the 52-untranslated region of mRNA transcripts that regulate gene expression. Binding of a specific small metabolite to the aptamer domain of the riboswitch causes structural changes that signal the formation of an alternative structure in the expression platform which in turns affects gene expression. Primarily found in bacteria, riboswitches have been shown to control many important and essential genes in virulent pathogens. However, the mechanism of coupling conformational changes within the aptamer domain to downstream structure formation is unknown. The goal of this work is to characterize the spatial and temporal structural changes that occur within the aptamer domain of the B12 riboswitch using standard biochemical techniques. In Aim 1, the local structural changes to the aptamer domain will be investigated using chemical probing techniques that will highlight the importance of magnesium and adenosylcobalamin (AdoCbl) to promote a stable structure. Also, solution X-ray scattering of wild type and mutant B12 riboswitches will help define helical arrangement and packaging in solution and changes upon AdoCbl binding. In Aim 2, time-resolved hydroxyl radical footprinting will investigate the rate of aptamer folding in the presence of magnesium and if AdoCbl aids this process. Using co-transcriptional time-resolved footprinting, the importance of the aptamer domain folding will be investigated under the timeframe of native transcription. Finally, to understand how ligand binding is conveyed to the expression platform, rates of RNA folding of the entire riboswitch with and without AdoCbl will be determined. The results of these experiments will increase the general knowledge of riboswitch structure formation as well as begin to dissect the mechanism of gene regulation. PUBLIC HEALTH RELEVANCE: Riboswitches are RNA elements that control gene expression primarily in bacterial cells through a small metabolite-dependent fashion. As more bacterial strains become resistant to current antimicrobial drugs, a deeper understanding of how riboswitches work will open up new possibilities of antimicrobial therapeutics.